Nitrate therapy for peripheral hypertension (HTN) is limited in efficacy due to the rapid induction of tachyphylaxis, via its inactivation of mitochondrial aldehyde dehydrogenase. The introduction of a nitrate therapy free of tolerance induction would represent a significant commercial opportunity and address a major unmet medical need. Radikal Therapeutics (RTX) is developing R-100, a small molecule agent formed from the covalent linkage of: 1) an organic nitrovasodilator domain that releases nitric oxide (NO), and 2) a pyrrolidine nitroxide domain that acts as a catalyst of reactive oxygen species degradation: a superoxide dismutase (SOD) mimetic, a catalase mimic that detoxifies hydrogen peroxide, and a peroxynitrite decomposition catalyst. R-100 has been proven in ex vivo and in vivo rodent systems to act as a powerful vasodilator that does not induce tolerance. R-100 has not yet been evaluated for induction of nitrate tolerance in chronically-dosed conscious large animals, a critical milestone in its pharmaceutical development. Aim #1: Establish the pharmacodynamic (PD) profile of R-100 in a conscious, ambulatory, large-animal model of HTN A series of hemodynamic studies wil be conducted in conscious, instrumented, ambulatory, and telemetered minipigs with HTN secondary to an endovascular-created, unilateral, renovascular stenosis. In Series A, we will correlate the pharmacokinetic (PK) profile and hemodynamic effect of single administration of R-100 (3, 10, 20, 30 mg/kg), with a 1-week washout period between successive dose levels. Animals will be monitored for 24 h to collect continuous hemodynamic data and intermittent plasma concentrations of R-100 and its metabolites. In Series B, we will evaluate the most promising 2 doses of R-100 from Series A, by carrying out a repeat-dose study of R-100 administered q6h for a period of 3 days. The hemodynamic effects of R-100 will be correlated with plasma peak and trough concentrations of R-100 and its metabolites obtained q6h. In Series C, we will evaluate the emergence of tachyphylaxis to R-100 in a 2-week repeat-dose study (q6h) utilizing the optimal dose determined in Series B, wherein hemodynamics will be correlated with plasma PK determinations as in Series B. Taken together, these studies will establish in a large animal model: 1) the relevant dose range, 2) the PK profile, 3) the PD profile, and 4) the extent of tolerance. Aim #2: Establish the in vitro metabolic profile of R-100 induced by exposure to human liver and intestinal microsomes. Prominent metabolites will be synthesized and evaluated for secondary metabolism in human microsomes. These findings will be further refined by identifying human CYPP450 enzymes that are responsible for metabolic transformation of R-100. Lastly, we will determine if R-100 inhibits major human CYP450 enzymes. The results of the proposed studies are expected to serve as the basis for a Phase 1a IND application to the FDA examining the safety and efficacy of oral R-100 in subjects with stable essential HTN. Follow-on Phase 1b repeat-dose 2-week studies will additionally establish whether R-100 induces tolerance in man. PUBLIC HEALTH RELEVANCE: Refractory peripheral hypertension is a frequent and life-threatening condition that increases the risk of cardiovascular events such as stroke and aortic aneurysm. We are developing a novel drug that targets the basic mechanisms of refractory hypertension and has been shown to be effective in pilot studies in a small animal model. We now propose to define the value of our technology in a definitive clinically-relevant large animal model of hypertension.